Fungal Keratitis Workup

Updated: Jul 19, 2019
  • Author: Michael Ross, MD; Chief Editor: Hampton Roy, Sr, MD  more...
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Laboratory Studies

The diagnosis of fungal keratitis continues to be problematic. Many clinical characteristics are not specific to fungal ulcers; therefore, antifungal therapy should be withheld until a diagnosis is confirmed by laboratory studies.

The most important step in the initial management of suspected fungal keratitis is to obtain corneal material for directed smears and inoculation of media. Smears are used to obtain rapid information about the pathogen. Gram stain identifies yeast, and Giemsa stain is useful in detecting fungal elements. However, if fluorescein microscopy is available, acridine orange and calcofluor white are the stains of choice. The primary isolation cultures for fungus are Sabouraud and blood agar at room temperature.

In all patients with suspected fungal keratitis, initial corneal smears and cultures should be performed. Culture media for suspected fungal keratitis should include the same media used for a general infectious-keratitis workup.

  • Corneal scrapings are obtained using a platinum spatula, surgical blade, or calcium alginate swab inoculated on Sabouraud agar plates, and then maintained at 25°C to enhance fungal growth.

  • Brain-heart infusion broth is another medium that can be used.

  • Cycloheximide should not be present in the medium because it inhibits fungal growth.

  • Corneal scrapings provide for initial debridement of organisms and epithelium, which may be a barrier to antifungal penetration.

  • Gram and Giemsa stains of corneal scrapings have sensitivities of about 50% in establishing a diagnosis. Calcofluor white stain (requires a fluorescent microscope) also can identify fungal organisms.

  • Initial growth in culture occurs within 72 hours in 83% of cultures and within 1 week in 97% of cultures.

  • A waiting period of 2 weeks is usually necessary for confirmation of no growth in culture.

  • Culture has been used as the criterion standard to aid ophthalmologists in the diagnosis of fungal keratitis; therefore, the true sensitivity of culture techniques is unknown.

When confronted with a likely fungal ulcer of days or weeks duration, a clinician in a developing country must urgently institute an antifungal treatment regimen; however, the choice of treatment is limited based on the antifungal agents available in that particular market.


Imaging Studies

If clinical evidence or suspicion of posterior segment involvement exists, ophthalmic B-scan ultrasound may be necessary to rule out concurrent fungal endophthalmitis.


Other Tests

The laboratory diagnosis of fungal keratitis may be problematic because of the very small sample obtained by scraping the corneal ulcer. Therefore, recent methods for the identification of fungi have been under study and include immunofluorescence staining, electron microscopy, and confocal microscopy. Confocal microscopy may help in correctly diagnosing early stages of fungal keratitis and in monitoring disease progress at the edges and depth. It may also help guide timely decision for keratoplasty and may be helpful in determining when to stop medication. [2]

The polymerase chain reaction (PCR) technique holds promise as an effective method of diagnosing fungal keratitis because it offers increased sensitivity and significant reduction in the time required to establish a diagnosis.



If corneal smears and cultures are negative at 48-72 hours in a patient who is strongly suspected of having a fungal infection and who is not improving on the initial, broad-spectrum antibacterial therapy, the authors recommend proceeding to a corneal biopsy to establish a diagnosis.

The corneal biopsy specimen should be submitted to the laboratory. A substantial portion also should be submitted for histopathologic examination. The pathologist should be alerted regarding the suspected diagnosis and especially that the specimen is a small piece of cornea.

A negative biopsy result indicates that the destructive corneal process is progressing, and hypopyon exists; therefore, anterior chamber paracentesis or excisional biopsy (keratoplasty) should be performed.


Histologic Findings

Histopathologic examination of corneal buttons can reveal the presence of fungal elements in 75% of patients. Fungal hyphae usually lie parallel to the corneal surface and lamellae. The presence of vertical oriented fungal elements in regard to stromal lamellae depicts high virulence of the organism and usually is associated with more aggressive infection. The Descemet membrane may serve as a partial barrier for invasion of fungal organisms. Penetration of the Descemet membrane by the fungal elements depicts an aggressive organism and a higher risk for contamination of the globe.